Human Thymosin, beta15 gene, protein and uses thereof

ABSTRACT

The present inventors have now discovered that humans have a gene that encodes a novel protein of the thymosin β family. This novel protein, herein referred to as thymosin β 15, has the ability to bind and sequester G-actin, like other members of the thymosin β family, but unlike what is known about other members it also directly regulates cell motility in prostatic carcinoma cells. The present invention is direct to an isolated cDNA encoding the human thymosin β 15 gene (SEQ ID NO: 1) and have deduced the amino acid sequence (SEQ ID NO: 2).

BACKGROUND OF THE INVENTION

[0001] The present invention provides novel genes, proteins, and usesthereof including, methods for diagnosing and treating cancer,particularly metastatic cancer.

[0002] Most eukaryotic cells (execptions include red blood cells andadult muscles) contain high concentrations, i.e., up to ˜250,μmol/l, ofmomomeric actin. How such actin remains unpolymerized in the cytoplasmhas remained a problem in cell biology (Nachmiar, V., Current Opinion inCell Biology, 1993, 5:56). Profilin, originally thought to be theactin-sequestering protein, is not present in sufficient amounts toaccount for more than part of the monomeric actin levels observed.Recently, an actin-sequestering 5 kD peptide was discovered in highconcentration in human platelets (Safer, et al., Proc. Natl. Acad. SciUSA 1990 87:2536-2540) and shown to be identical to a previously knownpeptide (Safter, et al., J. Biol. Chem., 1991, 268:4029-4032) originallythought to be the thymic hormone, thymosin β₄ (Tβ₄) (D. Safer, J. MuscleRes. Cell Motil, 1 992. 1 3:269-271). A detailed kinetic study of theinteraction of Tβ₄ and actin (Weber, et al., Biochemistry 1992,31:6179-6185)), together with other studies (Yu, et al., J. Biol. Chem.,1993, 268:502-509 and Cassimelds, et al., J. Cell Biol., 1992,119:1261-1270) support the hypothesis that Tβ₄ and Tβ₁₀ function primaryas G-actin buffers. Unpublished data (E. Hannappel) extend the functionto several other βthymosins. Tβ₄ has also been shown to inhibitnucleotide exchange by actin, whereas profilin increases the rate ofexchange (Coldschmidt-Clermont, et al., Mol. Cell Biol., 1992,3:1015-1025).

[0003] All vertebrates studied contain one or often two β-thymosins.Thus, the members of the β-thymosin family are believed to be importantin all species. Three new family members (Low, et al., Arch. Biochem.Biophys., 1992, 293:32-39 and Schmid, B., Ph.D Thesis, University ofTubingen 1 989) have been found in perch, trout and in sea urchin, thefirst non-vertebrate source. The sequences are well conserved suggestinghat actin sequestration is probably a property of all β-thymosins.However, when Tβ₄ was discovered and its sequence first determined in1981 (Low, et al., Proc. Natl. Acad. Sci., USA 1981, 78:1162-1166), datawere presented that suggested two extracellular functions (Low, et al.supra and Rebar, et al., Science 1981, 214:669-671). Two recent papersindicate a different and unexpected effect of a tetrapeptide which maybe derived from the amino terminus of Tβ₄.

[0004] Several reports demonstrate regulation of Tβ₄ or Tβ₁₀ synthesisat the transcriptional or translational level. An interferon-induciblegene (Cassimelds, et al., J. Cell. Biol. 1992, 119:1261-1270 andSanders, et al., Proc. Natl. Acad. Sci. USA 1992, 89:4678-4682) isidentical to the cDNA of human Tβ₄, and there are several genes for Tβ₄in humans. (Clauss, et al., Genomies 1991, 9:75-180 and Gomez-Marquez,et al., J. Immunol. 1989, 143:2740-2744)

[0005] It would be desirable to identify new members of the β-thymosinfamily, particularly in humans.

[0006] Bao and Zetter reported in an abstract presented at the AmericanAssociation for Cancer Research annual meeting (March 18-22. 1995) thedifferential expression of a novel mRNA expressed in high-metastatic rattumor cell lines, but not in a low metastatic variant. cDNA was isolatedand was reported to encode a protein with 68% identity to the ratthymosin β4. However, the nucleotide sequence and the deduced amino acidsequence were not reported.

SUMMARY OF THE INVENTION

[0007] We have now discovered that humans have a gene that encodes anovel protein of the thymosin β family. This novel protein, hereinreferred to as thymosin β15, has the ability to bind and sequesterG-actin, like other members of the thymosin β family, but unlike what isknown about other members it also directly regulates cell motility inprostatic carcinoma cells. We have isolated a cDNA of the humanthymosin, β15 gene (SEQ ID NO: 1) and have deduced the amino acidsequence (SEQ ID NO: 2). We have shown that enhanced transcripts (mRNA)and expression of the thymosin β15 gene in non-testicular cells has ahigh correlation to disease state in a number of cancers, such asprostate, lung, melanoma and breast cancer, particularly metastaticcancers. Accordingly, discovering enhanced levels of transcript or geneproduct in non-testicular tissues can be used in not only a diagnosticmanner, but a prognostic manner for particular cancers.

[0008] The present invention provides isolated nucleic acids(polynucleotides) which encode thymosin β15 having the deduced aminoacid sequence of SEQ ID. NO: 2 or a unique fragment thereof. The term“unique fragment” refers to a portion of the nucleotide sequence orpolypeptide of the invention that will contain sequences (eithernucleotides or amino acid residues) present in thymosin β15 (SEQ ID NO:2) but not in other member of the thymosin family. This can bedetermined when the hybridization profile of that fragment understringent conditions is such that it does not hybridize to other membersof the thymosin family. Such fragments can be ascertained from FIG. 3. Apreferred set of unique fragments are those that contain, or containpolynucleotides that encode, amino acid 7 to 12 of SEQ ID NO: 2, aminoacid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ ID NO: 2.Preferably, the unique nucleotide sequence fragment is 10 to 60nucleotides in length, more preferably, 20 to 50 nucleotides, mostpreferably, 30 to 50 nbtides. Preferably, the unique polypeptidesequence fragment is 4 to 20 amino acids in length, more preferably, 6to 15 amino acids, most preferably, 6 to 1 0 amino acids.

[0009] The polynucleotides of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptides may beidentical to the coding sequence shown in SEQ ID NO: 1 or may be adifferent coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same proteinas the DNA of SEQ ID NO: 1.

[0010] The polynucleotide may have a coding sequence which is anaturally occurring allelic variant of the coding sequence shown in SEQID NO: 1. As known in the art, an allelic variant is an alternate formof a polynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded protein.

[0011] The present invention also provides an isolated polynucleotidesegment which hybridize under stringent conditions to a unique portionof the hereinabove-described polynucleotides, particularly SEQ ID NO:1.The segment preferably comprises at least 10 nucleotides. As hereinused, the term “stringent conditions” means hybridization will occuronly if there is at least 95% and preferably at least 97%.identitybetween the sequences. These isolated segments may be used in nucleicacid amplification techniques, e.g., PCR, to identify and/or isolatepolynucleotides encoding thymosin β15.

[0012] As used herein a polynucleotide “substantially identical” to SEQID NO:1 is one comprising at least 90% homology, preferably at least 95%homology, most preferably 99% homology to SEC ID NO: 1. The reason forthis is that such a sequence can encode thyinosin β15 in multiplemammalian species.

[0013] The present invention'further provides an isolated and purifiedhuman thymosin β15 having the amino acid sequence of SEQ2 ID NO:

[0014] 2, or a unique fragment thereof, as well as polypeptidescomprising such unique fragments, including, for exarmple, amino acid 7to 1 2 of 25 SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and aminoacid 36 to 45 of SEQ ID NO: 2.

[0015] In accordance with yet another aspect of the present invention,there are provided isolated antibodies or antibody fragments whichselectively binds human thymosin β15. The antibody fragments include,for example, Fab, Fab′, F(ab′)2 or Fv fragments. The antibody may be asingle chain antibody, a humanized antibody or a chimeric antibody.

[0016] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotides orpolypeptides present in a living animal is not isolated, but the samepolynucleotides or DNA or polypeptides, separated from some or all ofthe coexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0017] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0018] The present invention further provides a method of treating aneoplastic cell expressing human thymosin β15 by administering to thecell an effective amount of a compound which suppresses the activity orproduction of the human thymosin β15 Preferably, the compound interfereswith the expression of the human thymosin β15 gene. Such compoundsinclude, for example, antisense oligonucleotides, ribozymes, antibodies,including single chain antibodies and fragments thereof.

DESCRIPTION OF THE DRAWINGS

[0019]FIGS. 1A and 1B show differential mRNA display and Northernanalysis of Dunning R-3327 rat prostatic adenocarcinoma variants. TotalRNA from AT2.1 (lane 1), AT3.1 (lane 2) and AT6.1 (lane 3) cells werereverse- transcribed and amplified by PCR with a primer set, T₁₁ AG anda 10 mer AGGGAACGAG (SEQ ID NO:3) in the presence of [α35-S]dATP. ThePCR fragments were displayed on a 6% polyacrylamide gel andautoradiographed. The differentially expressed band is indicated byarrowhead. B. Northern blot analysis of thymosin β15 gene. Two jig ofpoly (A) RNA was isolated from Dunning R-3327 variants AT2. l (lane 1),AT3.1 (lane 2), AT6.1 (lane 3), and Mat Lylu (lane 4), fractionated on a1.1 % formaldehyde-agarose gel, transferred to Hybond-N +nylon membrane(Amersham) and hybridized with a random primed (Grillon C, et al., FEBS1990, 274:30-34) ³²P-labeled Tβ15 cDNA fragment. The same blot washybridized with a ratβ-actin probe to demonstrate that equivalentamounts of RNA were loaded in each lane.

[0020]FIG. 2 is the nucleotide sequence (SEQ,ID NO.: 1) of Tβ15 cDNA 20and the predicted amino acid sequence (SEQ ID NO.: 2) (single-lettercode). The sequence numbers of the nucleotides and amino acids areindicated on the right side of the sequences. The translation initiationcodon ATG is underlined, and the termination codon TAA is marked with anasterisk. A putative actin binding region is underlined. These sequencedata are available from GenBank under accession number U25684.

[0021]FIG. 3 shows the alignment of the deduced Tβ15 protein sequenceand some of the other βthymosin isoforms. Regions of amino acid identityare represented by white letters boxed in black. Unboxed black letterscorrespond to nonidentical regions. Dots correspond to gaps introducedin the sequence to optimize alignment.

[0022]FIG. 4 shows expression of Tβ15 in various rat tissues. Themuitiple-tissue blot was obtained from Clontech. The blot was hybridizedwith the Tβ15 cDNA probe. Rat GAPDH is a loading control.

[0023]FIGS. 5A and 5B show in situ hybridization with antisenseriboprobe for Tβ15 on prostatic adenocarcinoma patients. FIG. 5A showsdifferential expression in tumors. The small arrow shows positivestaining. The large arrow shows negative staining. FIG. 5B shows that inpoorly differentiated and invasive prostate carcinoma, single cellsinvading stroma display intense staining (arrow).

[0024]FIGS. 6A, 6B and 6C show the effect of Tβ15 on actinpolymerization.

[0025]FIG. 6A. 3μM of pyrene-labeled G-actin was polymerized in thepresence of various amounts of GST-Tβ4 fusion peptide (▾), GST-Tβ15 (▴)or GST alone (∘). The final extent of polymerization was determined fromthe final levels of pyrene-labeled actin (fluorescence). All solutionscontained 5.5 mM Tris, pH7.6, 167 μM CaCl₂, 0.5 mM glutathione, 167 μMDTT, and 420 μM ATP. Polymerization was induced by addition of 2 mMMgCI₂ and 150 mM KCl. Error bars denote the range of duplicatemeasurements made from separate dilutions of the fusion proteins.

[0026]FIG. 6B. 2 μM of pyrene-labeled G-actin was polymerized in thepresence of various amounts of monomeric Tβ15 that had been cleaved fromGST by thrombin. The relative rates of polymerization were derived fromthe maximal rate of fluorescence increase in the initial phase ofpolymerization.

[0027]FIG. 6C. The final extent of actin assembly was determined by thesame methods used for the thymosin GST fusion peptides.

[0028] Experimental conditions are those described for FIG. 6B.

[0029]FIGS. 7A, 7B and 7C show serum stimulated migration of controltransfected and Tβ15 transfected Dunning R-3327 variants and theirgrowth rate. FIG. 7A. Vector control transfected (∘,∇) and Tβ15antisense (∘,∇) transfected AT3.1 cell clones. FIG. 7B. Vector controltransfected (∘,∇) and Tβ15 sense transfected (∘,∇) AT2.1 cell clones.Data are expressed as the mean ±SE (n =4). FIG. 7C. Growth curves ofcontrol transfected and Tβ15 (sense or antisense) transfected DunningR-3327 clones. Cells from vector control transfected AT2.1 (∘), Tβ15sense transfected AT2.1 (∘), vector control transfected AT3.1 (∇) andTβ15 antisense transfected AT3.1(∇) were plated at initial 10⁴cells/well in RPMI 1640 with 10% FBS and 250 nM dexamethasome in 12-wellplates.. Cells were harvested and counted at indicated times. Pointsrepresent the mean ±SE (n =3).

[0030]FIGS. 8A and 8B show Western analysis of thymosin, β-GST fusionprotein. FIG. 8A is a Coomasie staining of GST-Tβ fusion proteins. FIG.8B is a Western analysis of GST-Tμ fusion proteins with affinitypurified anti-Tβ15 C-terminal peptide antibody. Lane 1:

[0031] GST-Tβ4; Lane 2: GST-Tβ15; Lane 3: GST only

[0032]FIG. 9 shows a Northern analysis of thymosin β15 in mouse lungtumor cells. LA-4: mouse lung adenoma cell line; M27 and H59: metastaticvariants derived from mouse Lewis lung adenocarcinoma cell line.Northern blot analysis revealed that the probe detected the thymosin β15mRNA expression in M27 cells, less expression in H59 cells, but noexpression in LA-4 cells.

[0033]FIG. 10A, 10B, 10C and 10 show immunohistochemical staining ofhuman prostatic carcinoma tissues with an affinity purified polyclonalantibody to thymosin β15. A. Nonmalignant prostatic epithelia (largearrow) and high-grade prostatic intraepithelial neoplasia (PIN) (smallarrow). B. Moderately differentiated prostatic carcinoma showingheterogeneoue immunosi aining (small arrow, positive; large arrow,negative). C. Poorly differentiated prostatic carcinoma. D. Single cellsinvading stroma showing intense staining.

[0034]FIG. 11 is a 1.4% agarose gel electrophoresis of RT-PCR amplifiedβthymosins from the rat prostatic cell lines. Lane 1, weakly metastaticAT2.1; lane 2, 3 and 4, highly metastatic AT3.1, AT6.1 and Mat Lylu;lane 5 and 6, nonmetastatic NbE and MC2; lane 7, weakly metastatic Fb2.β-actin PCR was used as internal control of each sample.

DETAILED DESCRIPTION OF THE INVENTION

[0035] A well characterized series of cell lines that show varyingmetastatic potential has been developed from the Dunning rat prostaticcarcinoma (Isaacs, et al., Prostate 9, 261-281 and Bussebakers, et al.,Cancer Res. 52,2916-2922 (1992)). Coffey and colleagues previouslyshowed a direct correlation between cell motility and metastaticpotential in the Dunning cell lines (Mohler, et al., Cancer Res. 48,4312-4317 (1988), Parin, et al., Proc. Natl. Acad. Sci, USA 86,1254-1258 (1989) and Mohler, et al., Cancer Metast. Rev 12, 53-67(1993)). We compared gene expression in poorly metastatic and highlymetastatic cell lines derived from Dunning rat prostate carcinoma usingdifferential mRNA display. The results of these studies revealed theexpression of a novel member of the thymosin beta family of 5actin-binding molecules, thymosin β15. Using this information, weisolated and sequenced a cDNA encoding human thymosin β15.

[0036] Although members of the thymosin β family have been shown to bindand sequester G-actin, they have not previously been demonstrated toalter cell motility. Our studies, however, reveal that this new member,thymosin β15, directly regulates cell motility in prostatic carcinomacells. We have shown that expression of thymosin β15 is upregulated inhighly metastatic prostate cancer cell lines relative to poorlymetastatic or nonmetastatic lines. In addition, thymosin β15 wasexpressed in human prostate carcinoma specimens but not in normal humanprostate. Although not wishing to be bound by theory, this indicatesthat β15 plays a role in the process of metastatic transformation.

[0037] The present invention provides a polynucleotide sequence encodingall or part of thymosin β15 having the deduced amino acid sequence ofSEQ ID NO:2 or a unique fragment thereof. A-nucleotide sequence encodinghuman thymosin β15 is set forth as SEQ ID NO:1.

[0038] The sequences of the invention may also be engineered to providerestriction sites, if desired. This can be done so as not to interferewith the peptide sequence of the encoded thymosin β15, or may interfereto any extent desired or necessary, provided that the final product hasthe properties desired.

[0039] Where it is desired to express thymosin β15 or a unique fragmentthereof, any suitable system can be used. The general nature of suitablevectors, expression vectors and constructions therefor will be apparentto those skilled in the art.

[0040] Suitable expression vectors may be based on phages or plasmids,both of which are generally host-specific, although these can often beengineered for other hosts. Other suitable vectors include cosmids andretroviruses, and any other vehicles, which may or may not be specificfor a given system. Control sequences, such as recognition, promoter,operator, inducer, terminator and other sequences essential and oruseful in the regulation of expression, will be readily apparent tothose skilled in the art, and may be associated with the naturalthymosin β15 or with the vector used, or may be derived from any othersource as suitable. The vectors may be modified or engineered in anysuitable manner.

[0041] Correct preparation of nucleotide sequences may be confirmed, forexample, by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA74:5463-7 (1977)).

[0042] A cDNA fragment encoding the thymosin β15 of the invention mayreadily be inserted into a suitable vector. Ideally, the receivingvector has suitable restriction sites for ease of insertion, butblunt-end ligation, for example, may also be used, although this maylead to uncertainty over reading frame and direction of insertion. Insuch an instance, it is a matter of course to test transformants forexpression, 1 in 6 of which should have the correct reading frame.Suitable vectors may be selected as a matter of course by those skilledin the art according to the expression system desired.

[0043] By transforming a suitable organism or, preferably, eukaryoticcell line, such as HeLa, with the plasmid obtained, selecting thetransformant with ampicillin or by other suitable means if required, andadding tryptophan or other suitable promoter-inducer (such asindoleacrylic acid) if necessary, the desired thymosin β15 may beexpressed. The extent of expression may be analyzed by SDSpolyacrylamide gel electrophoresis-SDS-PAGE (Lemelli, Nature 227:680-685(1970)).

[0044] Suitable methods for growing and transforming cultures etc. areusefully illustrated in, for example, Maniatis (Molecular Cloning, ALaboratory Notebook, Maniatis et al. (eds.), Cold Spring Harbor Labs,N.Y. (1989)).

[0045] Cultures useful for production of thymosin β15, or a peptidethereof, may suitably be cultures of any living cells, and may vary fromprokaryotic expression systems up to eukaryotic expression systems. Onepreferred prokaryotic system is that of E. coli, owing to its ease ofmanipulation. However, it is also possible to use a higher system, suchas a mammalian cell line, for expression of a eukaryotic protein.Currently preferred cell lines for transient expression are the HeLa andCos cell lines. Other expression systems include the Chinese HamsterOvary (CHO) cell line and the baculovirus system.

[0046] Other expression systems which may be employed includestreptomycetes, for example, and yeasts, such as Saccharomyces spp.,especially S. cerevisiae. Any system may be used as desired, generallydepending on what is required by the operator. Suitable systems may alsobe used to amplify the genetic material, but it is generally convenientto use E. coli for this purpose when only proliferation of the DNA isrequired.

[0047] Standard detection techniques well known in the art for detectingRNA, DNA, proteins and peptides can readily be applied to detectthymosin β15 or its transcript to diagnose cancer, especially metastaticcancer or to confirm that a primary tumor has, or has not, reached aparticular metastatic phase.

[0048] In one such technique, immunohistochemistry, anti-thymosin β15antibodies may be used to detect thymosin β15 in a biopsy sample.

[0049] Anti-thymosin β15 antibodies may also be used for imagingpurposes, for example, to detect tumor metastasis. Suitable labelsinclude radioisotopes, iodine (¹²⁵l, ¹²¹l), carbon (¹⁴C), sulphur (³⁵S),tritium (³H), indium (¹¹²ln), and technetium (⁹⁹mTc), fluorescentlabels, such as fluorescein and rhodamine, and biotin.

[0050] However, for in vivo imaging purposes, the position becomes morerestrictive, as antibodies are not detectable, as such, from outside thebody, and so must be labelled, or otherwise modified, to permitdetection. Markers for this purpose may be any that do not substantiallyinterfere with the antibody binding, but which allow external detection.Suitable markers may include those that may be detected byX-radiography, NMR or MIR. For X-radiographic techniques, suitablemarkers include any radioisotope that emits detectable radiation butthat is not overtly harmful to the patient, such as barium or caesium,for example. Suitable markers for NMR and MIR generally include thosewith a detectable characteristic spin, such as deuterium, which may beincorporated into the antibody by suitable labelling of nutrients forthe relevant hybridoma, for example.

[0051] In the case of in vivo imaging methods, an antibody or antibodyfragment which has been labelled with an appropriate detectable imagingmoiety, such as a radioisotope (for example, ¹³l, ¹²ln, 99m Tc), aradio-opaque substance, or a material detectable by nuclear magneticresonance, is introduced (for example, parenterally, subcutaneously orintraperitoneally) into the subject (such as a human) to be examined.The size of the subject, and the imaging system used, will determine thequantity of imaging moiety needed to produce diagnostic images. In thecase of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of technetium-99m. The labelled antibody or antibodyfragment will then preferentially accumulate at the location of cellswhich contain thymosin β15 The labelled antibody or antibody fragmentcan then be detected using known techniques.

[0052] The antibodies may be raised against either a peptide of thymosinβ15 or the whole molecule. Such a peptide may be presented together witha carrier protein, such as an KLH, to an animal system or, if it is longenough, say 25 amino acid residues, without a carrier. Preferredpeptides include regions unique to thymosin β15, such as amino acid 7 to12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid36 to 45 of SEQ ID NO: 2.

[0053] Polyclonal antibodies generated by the above technique may beused direct, or suitable antibody producing cells may be isolated fromthe animal and used to form a hybridoma by known means (Kohler andMilstein, Nature 256:795. (1975)). Selection of an appropriate hybridomawill also be apparent to those skilled in the art, and the resultingantibody may be used in a suitable assay to identify thymosin β15.

[0054] Antibodies, or their equivalents, may also be used in accordancewith the present invention for the treatment or prophylaxis of cancers.Administration of a suitable dose of the antibody may serve to blockproduction, or to block the effective activity of thymosin β15, and thismay provide a crucial time window in which to treat the malignantgrowth.

[0055] Prophylaxis may be appropriate even at very early stages of thedisease, as it is not known what actually leads to metastasis in anygiven case. Thus, administration of the antibodies, their equivalents,or factors which interfere with thymosin β15 activity, may be effectedas soon as cancer is diagnosed, and treatment continued for as long asis necessary, preferably until the threat of the disease has beenremoved. Such treatment may also be used prophylactically in individualsat high risk for development of certain cancers, e.g., prostate.

[0056] A method of treatment involves attachment of a suitable toxin tothe antibodies which then target the area of the tumor. Such toxins arewell known in the art, and may comprise toxic radioisotopes, heavymetals, enzymes and complement activators, as well as such naturaltoxins as ricin which are capable of acting at the level of only one ortwo molecules per cell. It may also be possible to use such a techniqueto deliver localized doses of suitable physiologically active compounds,which may be used, for example, to treat cancers.

[0057] It will be appreciated that antibodies for use in accordance withthe present invention, whether for diagnostic or therapeuticapplications, may be monoclonal or polyclonal as appropriate. Antibodyequivalents of these may comprise: the Fab' fragments of the antibodies,such as Fab, Fab', F(ab′)2 and Fv; idiotopes; or the results of allotopegrafting (where the recognition region of an animal antibody is graftedinto the appropriate region of a human antibody to avoid an immuneresponse in the patient), for example. Single chain antibodies may alsobe used. Other suitable modifications and/or agents will be apparent tothose skilled in the art.

[0058] Chimeric and humanized antibodies are also within the scope ofthe invention. It is expected that chimeric and humanized antibodieswould be less immunogenic in a human subject than the corresponding 15non-chimeric antibody. A variety of approaches for making chimericantibodies, comprising for example a non-human variable region and ahuman constant region, have been described. See, for example, Morrisonet al., Proc. Natl. Acad. Sci. U.S.A. 81,6851“(1985); Takeda et al.,Nature 314,452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; 20 Bosset al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP 171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B. Additonally, a chimeric antibody can befurther “humanized” such that parts of the variable regions, especiallythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such altered immunoglobulin molecules may be made by any of severaltechniques known in the art, (e.g., Teng et al., Proc. Natl. Acad Sci.U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279(1983); Oisson et al., Meth. Enzymol., 92, 3-16 (1982)), and arepreferably made according to the teachings of PCT Publication W092/06193or EP 0239400. Humanized antibodies can be commercially produced by, forexample, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, GreatBritain. 5 Another method of generating specific antibodies, or antibodyfragments, reactive against thymosin β15 is to screen phage expressionlibraries encoding immunoglobulin genes, or portions thereof, with aprotein of the invention, or peptide fragment thereof. For example,complete Fab fragments, V H regions and V-region 10 derivatives can beexpressed in bacteria using phage expression libraries. See for exampleWard, et al., Nature 341,544-546: (1989); Huse, et al., Science 246,1275-1281 (1989); and McCafferty, et al., Nature 348, 552-554 (1990).

[0059] The antibody can be administered by a number of methods. Onepreferred method is set forth by Marasco and Haseltine in PCTW094/02610, which is incorporated herein by reference. This methoddiscloses the intracellular delivery of a gene encoding the antibody, inthis case the thymosin β15 antibody. One would preferably use a gene 20encoding a single chain thymosin β15 antibody. The antibody wouldpreferably contain a nuclear localization sequence, for examplePro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO:4) (Lawford, et al. Cell 46:575(1986)]; Pro-Glu-Lys-Lys-lle-Lys-Ser (SEQ ID NO:5) (Stanton, et al.,Proc. Nat?. Acad. Sci. USA 83:1772 (1986)1, GIn-Pro-Lys-Lys-Pro (SEQ 25ID NO:6) (Harlow, et al., Mol. Cell. Bio/. 5:1605 (1985)1;Arg-Lys-Lys-Arg (SEQ ID NO:7) for the nucleus. One preferably uses anSV40 nuclear localization signal. By this method one can intracellularlyexpress a thymdsin β15 antibody, which can block thymosin β15functioning in desired cells.

[0060] In addition to using antibodies to inhibit thymosin β15, it mayalso be possible to use other forms of inhibitors. Inhibitors ofthymosin β15 may manufactured, and these will generally correspond tothe area of the substrate affected by the enzymatic activity. It isgenerally preferred that such inhibitors correspond to a frozenintermediate between the substrate and the cleavage products, but it isalso possible to provide a sterically hindered version of the bindingsite, or a version of the binding site which will, itself, irreversiblybind to thymosin β15 Other suitable inhibitors will be apparent to theskilled person.

[0061] The invention also provides for the treatment of a cancer byaltering the expression of the thymosin β15 This may be effected byinterfering with thymosin β15 production, such as by directing specificantibodies against the protein, which antibodies may be further modifiedto achieve the desired result. It may also be possible to block thethymosin β15 receptor, something which may be more easily achieved bylocalization of the necessary binding agent, which may be an antibody orsynthetic peptide, for example.

[0062] Affecting thymosin β15 gene expression may also be achieved moredirectly, such as by blocking of a site, such as the promoter, on thegenomic DNA.

[0063] Where the present invention provides for the administration of,for example, antibodies to a patient, then this may be by any suitableroute. If the tumor is still thought to be, or diagnosed as, localized,then an appropriate method or administration may be by injection directto the site. Administration may also be by injection, includingsubcutaneous, intramuscular, intravenous and intradermal injections.

[0064] Formulations may be any that are appropriate to the route ofadministration, and will be apparent to those skilled in the art. Theformulations may contain a suitable carrier, such as saline, and mayalso comprise bulking agents, other medicinal preparations, adjuvantsand any other suitable pharmaceutical ingredients. Catheters are anotherpreferred mode of administration.

[0065] Thymosin β15 expression may also be inhibited in vivo by the useof antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. An antisense nucleic acid molecule which is complementary to anucleic acid molecule encoding thymosin β15 can be designed based uponthe isolated nucleic acid molecules encoding thymosin β15 provided bythe invention. An antisense nucleic acid molecule can comprise anucleotide sequence which is complementary to a coding strand of anucleic acid, e.g. complementary to an mRNA sequence, constructedaccording to the rules of Watson and Crick base pairing, and canhydrogen bond to the coding strand of the nucleic acid. The antisensesequence complementary to a sequence of an mRNA can be complementary toa sequence in the coding region of the mRNA or can be complementary to a5′or 3′ untranslated region of the mRNA. Furthermore, an antisensenucleic acid can be complementary in sequence to a regulatory region ofthe gene encoding the mRNA, for instance a transcription initiationsequence or regulatory element. Preferably, an antisense nucleic acidcomplementary to a region preceding or spanning the initiation codon orin the 3′ untranslated region of an H. et al., Antisense RNA as amolecular tool for genetic analysis, Reviews - Trends in Genetics, Vol.1 (1)1986.

[0066] In addition, ribozymes can be used to inhibit in vitro expressionof thymosin β15. For example, the nucleic acids of the invention canfurther be used to design ribozymes which are capable of cleaving asingle-stranded nucleic acid encoding a β15 protein, such as a thymosinβ15 mRNA transcript. A catalytic RNA (ribozyme) having ribonucleaseactivity can be designed which has specificity for an mRNA encodingthymosin β15 based upon the sequence of a nucleic acid of the invention(e.g., SEQ ID NO: 1 ). For example, a derivative of a Tetrahymena L-19IVS RNA can be constructed in which the base sequence of the active siteis complementary to the base sequence to be cleaved in a thymosinβ15-encoding mRNA. See for example Cech, et al., U.S. Pat. No.4,987,071; Cech, et al., U.S. Pat. No. 5,116,742. Alternatively, anucleic acid of the invention could be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See for example Bartel, D. and Szostak, J. W. Science 261,1411-1418(1993).

[0067] Methods for the diagnosis and prognosis of cancer using thepolynucleotides and antibodies of the present invention are set forth incopending application (Docket No. 46403) Express Mail No. TB338582354US,the disclosure of which is herein incorporated by reference.

[0068] All references cited above or below are herein incorporated byreference.

[0069] The following Examples serve to illustrate the present invention,and are not intended to limit the invention in any manner.

EXAMPLES

[0070] METHODS

[0071] Cell Culture

[0072] The poorly metastatic AT2.1 subline and high metastatic AT3.1,AT6.1 and Mat lylu sublines derived from Dunning R3327 rat prostaticadenocarcinoma cells (provided by Dr. J. Issaacs, The Johns HopkinsUniversity) were maintained in vitro in RPMI 1640 medium, supplementedwith 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1 %glutamine/penicillin, streptomycin (Irvine Scientific, Santa Ana, CA),and 250 nM dexamethasome (Sigma Chemical Co, St. Louis, MD), under anatmosphere of 5% CO₂; 95% air at 37° C.

[0073] RNA Isolation and Northern Blot Analysis

[0074] Cells at 70% confluency were harvested and subjected to RNAisolation. Total RNA was prepared by acid guanidiniumthiocyanate/phenol/chloroform extraction procedures. (Chomczynski, P. &Sacchi, Anal. Biochem. 167, 157-159 (1987)). Poly (A) RNAs were isolatedfrom total RNA using Poly (A) Quik mRNA Isolation Kit (Stratagene, LaJolla, CA) or Micro Fast Track mRNA Isolation Kit (Invitrogen, SanDiego, CA). 20 μg of total RNA or 2 μg of mRNA was size fractionated ona denaturing formaldehyde agarose gel (1.1 %) and transferred ontoHybond-N-membrane (Amersham Corporation, Arlington Heights, IL) bycapillary blotting in 0.05 M NaOH buffer according to the manufacturer'sprocedure. Northern blot filters were prehybridized for 3 hours at 42°C. in 5x Denhardt's, 50% formamide, 5x SSPE, 0.5% SDS solutioncontaining 100 μg/ml denatured salmon sperm DNA (Stratagene), followedby overnight hybridization in fresh prehybridization solution with theaddition of denatured probe labeled with [alpha-³P] dCTP (New EnglandNuclear, Wilmington, DE) using random primed DNA labeling kit(Boehringer Mannheim Biochemica, Indianapolis, IN). Filters were washedat increasing stringency to a final stringency of 0.2 x SSC; 0.1 % SDSat 55° C. Autoradiography was performed over two days at −80° C. usingKodak X-Omat's film with intensifying screen. For reprobing, theoriginal probe was removed by the blots with boiling in 0.5% SDS waterfor 10 min.

[0075] mRNA differential display

[0076] DNase I digested 2μg of total RNA from AT2.1, AT3.1 and AT6.1cells grown to 70% confluency in RPMl 1640 medium supplemented with 10%fetal bovine serum and 250 nM dexamethasone were reverse-transcribedwith 300 units of MMLV reverse transcriptase (Stratagene) in thepresence of 2.5 μM of T 1 1 AG as primer and 20 μM dNTP for 60 min at35° C. After heat inactivation of the reverse transcriptase at 95° C.for 5 min, 2 μl of the sample was amplified by PCR with T11 AG primerand arbitrary 10 mers in the presence of [a-³⁵S]dATP (New EnglandNuclear). The PCR parameters were 94° C. for 30 sec, 42° C. for 1 min,and 72° C. for 30 sec for 40 cycles, followed by 5 min elongation at 72°C. PCR products were fractionated on a 6% polyacrylamide gel andvisualized by autoradiography. Differentially expressed bands were cutout of the dried gels and reamplified by PCR using the correspondingsets of primers. The reamplified PCR fragments were used as probes forNorthern blot analysis.

[0077] cDNA library screening

[0078] An oligo(dT)-primed cDNA library was constructed in the lambdagt10 vector (Amersham) using polyadenylated [poly(A)⁻] RNA obtained fromAT3.1 cells in culture. The library was screened with a ³²P-labeledprobe generated by PCR, using a 343 base pair AT3.1 cDNA isolated fromdifferential display as template. Filters were hybridized with probeovernight at 65° C. in a 5x Denhardt's, 5x SSPE, 0.5% SDS solutioncontaining 100μg/ml denatured salmon sperm DNA, and washed at highstringency with 0.2 x saline sodium citrate (SSC) and 0.1% SDS at 65° C.Inserts of positive clones were excised from Agt10 vector with EcoRlenzyme, subcloned into pbluescript 11 SK+/−(Stratagene) and sequencedusing the Sequenase Version 2.0 sequencing kit (U.S. Biochemical,Cleveland, OH).

[0079] RT-PCR Analysis

[0080] Total RNA from each cell line was digested with RNase free DNasel (GIBCO BRL, Gaithersburg, MD). DNase l digested 5,μg of total RNA wasreverse transcribed using cDNA Cyling Kit (invitrogen). The reversetranscrition mixture was purified with a Spin Column 300 (Pharmocia,Piscataway, NJ). 10,ul of purified cDNA was amplified with primer setsof Tβ15 forward primer: 5′-TATCAGCTAGTGGCTGCACCCGCG-3′ (SEQ ID NO:8) andreverse primer: 5′-AAATGCTGACCTTTCAGTCAGGGT-3′(SEQ ID NO:9); Tβ4 forwardprimer: 5′-ACTCTCAATTCCACCA TCTCCCAC-3′(SEQ ID NO:10), reverse primer:5′-GCCTCTGAGCAGATCGTCTCTCCT7G-3′(SEQ ID NO:11); and TBβ10 forwardprimer: 5′-ATAATATCCCTGGGCAAACCGGTG-3′(SEQ ID NO:12), reverse primer:5′-GAGTGGAG TACCTGGAGCGCGAGC-3′(SEQ ID NO:13), respectively. PCRamplification was performed in 50 pI of PCR reaction buffer (50 mM KCI,10 mM Tris [pH 8.51, 1.5 mM MgCI₂) with 1 mM of dNTPs, 50 pmol of eachprimer, and 2.5 U of Taq polymerase (GIBCO BRL), overlaid with 50 μl ofmineral oil (Sigma). The PCR profile was 94° C., 30 sec; 60° C., 30 sec;and 72° C., 2 min for 30 cycles. Control studies of the RT-PCR wereconducted using aliquats from the same samples and amplified withprimers to the β-actin gene (Clontech, Palo Alto, CA). Amplificationproducts were separated on 1.4% agarose gels.

[0081] In situ hybridization Antisense and sense Tβ15 mRNA probes wereprepared using Tβ15 cDNA inserted into the eukaryotic expression vectorpcDNA3 (Invitrogen) as template and a digoxigenin RNA labeling kit(Boehringer Mannheim). Formalin-fixed paraffin-embedded sections weredewaxed, rehydrated, and digested with proteinase K (50 pg/ml) in 100 mMTris, 50 mM EDTA buffer (pH 8) for 8 min at 37° C. Hybridization wasperformed in an automated instrument (Ventana Medical Systems, Tuscon,AZ) for 60 min at 42° C. with 10 βM digoxigenin-labeled riboprobe in 100pIl of hybridization buffer (50% deionized formamide, 4x SSC, 10%dextran sulfate, 1 % SOS, and denatured herring sperm DNA (400 pg/ml))per section under a liquid cover slip. The highest stringency ofposthybridization washes was at 45° C. for 15. min in 0.1 x SSC. Bounddigoxigenin-labeled probe was detected by anti-digoxigenin alkalinephosphatase conjugate and visualized by nitroblue tetrazolium and5-bromo-4chloro-3-indolylphosphate (NBT-BCIP) color reaction. Sectionswere counterstained with nuclear fast red.

[0082] GST-Tβ fusion protein expression

[0083] PCR generated DNA fragments containing the full coding regions ofTβ15 and Tβ4 were ligated in frame into the BamHI-EcoRl site of theprokaryotic expression vector pGEX-2T (Pharmacia, Piscataway, NJ). ThepGEX-Tβ fusions were expressed in Escherichia coli, strain DH5a, byincubating with 0.1 mM isopropylthio-β-D-galactoside for 3 hours. Cellswere recovered by centrifugation, washed, and suspended in Technica,Durham, NC). The migration of 5,000 cells placed in the upper welltoward fetal bovine serum in the lower well was assayed following a 4hour incubation at 37 C. After removal of cells from th upper side ofthe filters, cells that had passed through the filters and 5 adhered tothe lower side were fixed in formalin, washed with PBS an stained withGill's triple strength hematoxylin (Polysciences, Warrington, PA) andcounted under light microscopy. Generation of polyclonal antibody 100.25 mg of a synthetic oligopeptide (IQQEKEYNQRS) representing the 11carboxyl terminal amino acids of thymosin β15 dissolved in 380 ml of a0.125 M phosphate buffer, pH 7.4 was pipetted into reaction vesselcontaining 1.0 mg of keyhole limpet hemocyanin (Sigma). Then, 20,ul of25% aqueous glutaraldehyde 15 solution was added. After gentleagitation, first for 3 h at room temperature and then for 1 2 h at 4°C., the reaction mixture was diluted with 0.15 M Nail to a finalconcentration of 100 βg/ml. The diluted mixture was then used forimmunization. New Zealand White rabbits were immunized with 30 βg of theC-terminal peptide of thymosin β15 as KLH conjugate emulsified with CFA.The first booster injection was given 6 weeks after the firstimmunization. Whereas subsequent booster injections were given at 3weeks intervals. Production bleeds were obtained 2 weeks after the fifthboost. Antisera were affinity purified over the C-terminal peptideconjugated CNBr-activated Sepharose 4B column (Pharmacia) in 10 mMTris-HCI, pH 7.4. After extensive washing of the column with 0.5 M NaCl,10 mM Tris, pH 7.4, the column was eluted with 0.2 M Glycine, 0.2 NaCl,pH 2.0. The purity and specificity of eluted fractions were examined byWestern analysis.

[0084] Western analysis

[0085] GST-T, fusion proteins were run on a 12% SDS-polyacrylamide geland transfered to a nitrocellulose membrane (0.2mm, Schleicher &Schuell, Keene, NH). The blot was incubated with 5% nonfat dry milk inphosphate-buffered saline containing 0.1% Tween 20 (TBS-T) followed byincubation with the 1: 1000 diluted affinity purified anti Tβ15C-terminal peptide antidody for 1 h and washed 3 times with TBS-T. Theblot was then incubated with horseradish peroxidase-conjugatedanti-rabbit IgG antibody (Amersham Corp.) for 40 min, and a specificantibody reaction was detected by an enhanced chemiluminescencedetection system (Amersham Corp.).

[0086] Immunohistochemical staining

[0087] Human prostate cancer sections were studied using animmunoperoxidase ABC kit (Vector, Burlingame, CA). Briefly, the 5 βmtissue sections were deparaffinized in xylene, rehydrated in gradedalcohols, and blocked for endogenous peroxidase by 3% hydrogen peroxide(Sigma) in methanol for 30 min. The sections-were treated with normalgoat serum for 30 min and then incubated with an affinity purified antiTβ15 C-terminal peptide antibody for 2 h at room temperature at 1:100(v/v) dilution, followed by incubation with a biotinylated goatanti-rabbit IgG antibody for 30 min. After incubation with a preformedABC complex for 30 min, specifically bound antibodies were visualized byusing peroxidase substrate, 3, 3′-diaminobenzidine tetrahydrochloride(DAB). Sections were counterstained with Gill's hematoxylin.

RESULTS

[0088] Cloning of Tβ15

[0089] We compared patterns of gene expression by mRNA differentialdisplay analysis (Liang, P. & Pardee, A.B., Science 257, 967-971 (1992)in three variants of the Dunning rat tumor: the weakly metastatic,poorly motile line AT2.1 and the highly metastatic, highly motile linesAT3.1 and AT6.1. One band, which was detected in the more motile AT3.1and AT6.1 lines by differential display (FIG. 1A) was confirmed byNorthern (RNA) analysis to represent an overexpressed mRNA ofapproximately 420 nucleotides in AT3.1, AT6.1 as well as the relatedMatLyLu cell line but was not expressed in the poorly motile AT2.1 line(FIG. 1B). The gene was not expressed in other rat prostatic cell lines(non-metastatic) characterized by Northern analysis (data not shown).

[0090] To obtain a full-length complementary DNA (cDNA) clone of thisgene, an AT3.1 cDNA library was screened using the originally clonedcDNA fragment from differential display as a probe. A positive clonewith a 412 base pair insert was isolated, which contained a single open-reading frame encoding a 45 amino acid protein with a calculatedmolecular mass of 5304 (FIG. 2). The insert sizl-of the clone wasapproximately the same as the molecular size of the transcript seen inNorthern analysis suggesting that the clone contained the full lengthgene sequence. A computer assisted homology search against the Genebankand EMBL DNA databases revealed that the novel gene shared 49%nucleotide sequence homology with rat thymosins ,4 and 610. Alignment ofthe deduced amino acid sequence of the cloned gene with members of thethymosin , family (Mihelic, M. & Voelter, Amino Acids 6, 1-13 (1994)showed 68% homology with thymosin β4, 62% with thymosin P10 and 60% withβ9, β11 and β12 (FIG. 3). The results suggest that we have cloned anovel βthymosin, now named thymosin β15, from rat prostatic carcinomacells.

[0091] Hydropathy analysis of the thymosin β15 protein sequence revealedno apparent membrane-spanning or membrane-associated regions and noamino-terminal signal sequence. The protein is highly hydrophilic withan estimated isoelectric point of 5.14 and contains regions common toall members of the thymosin β family. All β-thymosin family memberspreviously studied, for example, have a putative actin binding region(LKKTET) 16 residues from the amino terminus (Vancompernolle, et al.,EMBO J. 11, 4739-4746 (1992), Troys, et al., EMBO J. 15, 201-210 (1996). Thymosin 15 also has such a region, although the glutamic acidresidue is replaced by an asparagine residue to form LKKTNT (FIG. 3).The principal region of nonconformity between members of the thymosin 6family occurs at the carboxyl terminus and the thymosin β15 sequence aswell shows no significant homology in this region with other familymembers.

[0092] Members of the 6-thymosin family may be independently expressedin different tissues (Un, et al., J. Biol. Chem. 266, 23347-23353(1991), Voisin, et al. J. Neurochem. 64, 109-120 (1995). Althoughthymosin,β15 is differentially expressed in the prostate carcinoma celllines tested, all of these lines expressed equivalent levels ofthymosins f4 and 81 0 by RT-PCR analysis (FIG. 1 1). The tissuedistribution of thymosin β15 mRNA was examined in the major organs ofthe rat. No expression of thymosin β15 was detected in the heart, brain,lung, spleen, liver, skeletal muscle and kidney, whereas high expressionwas found in the testis (FIG. 4). Southern (DNA) analysis of Hind IlIl-,EcoR I- and Pst I-restricted DNA from AT2.1 and AT3.1 cells withthymosin β15 cDNA probe revealed no gross structura; alteration of thethymosin β15 gene in the tumor cells (data not shown). These resultsdemonstrate that a novel member of the thymosin 8 family is upregulatedin metastatic rat prostatic carcinoma cell lines, whereas expression ofother thymosin β family members (β4 and β10) remains unchanged.

[0093] Cloning of Human Thymosin β15 by RT-PCR

[0094] DNase I digested 5 βmg of total RNA from human prostaticcarcinoma cell line PC-3 was,reverse transcribed using cDNA Cycling Kit(Invitrogen). The reverse transcription mixture was purified with a SpinColumn 300 (Pharmocia, Piscataway, NY). 10 βl of purified cDNA reactionwas amplified with primers Fl (5′-TATCAGCTAGTGGCTGCACCCGCG-3′) (SEQ IDNO:8) and RI (5′-AAATGCT GACCTTrCAGTCAGGGT-3′) (SEQ ID NO:9) designed toanneal to the outer ends of the thymosin β15 sequence. PCR amplificationwas performed in 50 βof PCR reaction buffer (50 mM KCI, 10 mM Tris (pH8.5], 1.5 mM MgCI2) with 1 mM of dNTPs, 50 pmol of each primer, and 2.5U of Taq polymerase (GItCO BRL), overlaid with 50 pI of mineral oil(Sigma). The PCR profile was 94° C., 30 sec; 60° C., 30 sec; and 72° C.,2 min for 30 cycles. Control studies of the RT-PCR were conducted usingaliquats from the same samples and amplified with primers to the,B-actingene (Clontech, Palo Alto, CA). Amplification products were separated on1.6% agarose gels. The amplified PCR product was ligated to pCR using TAcloning kit (Invitrogen, San Diego, (CA), and then DNA sequenced. Thesequence of the PCR product of human prostatic carcinoma cells amplifiedby the thymosin β15 primers was surprisingly 100% identical to thethymosin β15 sequence obtained from the rat prostatic carcinoma cells.Expression of Tβ15 mRNA in human prostat cancer

[0095] To determine whether this thymosin family member may be expressedin human prostate cancer, we examined human prostatic carcinoma cellline PC-3 by RT-PCR with forward and reverse primers for thymosin β15The PC-3 cells showed a low level of thymosin β15 expression. The DNAsequence of the amplified PCR product was 100% identical to the ratthymosin β15 sequence. We conducted in situ hybridization study onsamples from patients with varying grades of prostatic carcinomas usinga thymosin β15 probe. The tissue sections allowed direct comparison ofnormal and malignant elements on the same samples. The stromal elementswithin and around the tumor cell masses, as well as the nonmalignantprostatic epithelium adjacent to the tumor showed little backgroundhybridization with the thymosin β15 antisense probe. In contrast,specific tumor cell islands exhibited a strong specific thymosin β15signal when probed with antisense (FIG. 5A, small arrow) but not with asense RNA probe (data not shown). Although nearly all of the tumor cellsin the positive islands expressed thymosin β15 mRNA, not all patientspecimens were positive and not all islands in a single prostate werepositive (FIG. 5A, large arrow). The majority of the negative tumorcells were in 20 non-invasive in situ carcinomas whereas highly invasivetumors were consistently positive (FIG. 58). Thus a novel P thymosin,first detected in metastatic rat prostate carcinoma cell lines, isupregulated in human prostate cancer.

[0096] Effect of Tβ15 on actin polymerization

[0097] Because thymosin β15 retains a putative actin-binding domain, wetested its effect on actin polymerization using recombinant fusionproteins. The results, shown in FIG. 6A, reveal that aglutathione-S-transferase (GST)/thymosin β15 fusion protein inhibitspolymerization of pyrene-derivatized actin monomers to an equal orslightly greater extent than a GST/thymosin b4 fusion protein,suggesting that these two proteins have similar actin-sequesteringproperties. Similar results were obtained when thymosin,β15 was cleavedfrom the GST-fusion protein with thrombin and subsequently analyzed forits ability to inhibit the rate and extent of actin polymerization (FIG.6B and C). The difference in apparent affinity for actin between freeand GST-fused thymosin β15 is likely related to the GST-mediateddimerization of the fusion peptides to form complexes with two actinmonomer binding sites that either bind actin more tightly or bind to theend of the growing filament, thereby inhibiting polymerization at lowmolar ratios to total actin. One example of such an effect is the strongretardation of actin assembly by actobindin, which appears to functionas a dimer of thymosin-like actin binding sites (Bubb, et al.,Biochemistry 34, 3921-3926 (1995).

[0098] Effect of Tβ15 on cell motility

[0099] To determine whether thymosin β15 expression had an effect oncell motility, we transfected highly motile AT3.1 cells with aeukaryotic expression vector (pcDNA3) containing the thymosin β15 genein antisense orientation driven by the constitutive humancytomegalovirus promoter. The transfected cells growing in selective(G418) media were examined for expression of antisense transcripts ofthe thymosin β15 gene by strand-specific polymerase chain reaction (PCR)amplification (Zhou, et al., CancerRes. 52, 4280-4285 (1992). Analysisof cell motility in a multiwell Boyden chamber apparatus (Boyden, S. V.,J. Exp. Med. 115, 453-466 (1962)) using fetal bovine serum as amigration stimulus revealed that the motility of the transfectants whichshowed expression of antisense transcripts was significantly reducedrelative to the vector-only controls (FIG. 7A). Two antisensetransfected clones which did not express antisense transcripts failed toshow any decreased rate of cell motility (data not shown). In a furtherexperiment, poorly motile AT2.1 cells, transfected with sense thymosin.15 constructs and confirmed to express thymosin β15 by Northernanalysis, were shown to have significantly increased stimulated motilityrelative to their vector controls (FIG. 7B). Both the sense andantisense thymosin β15 transfectants showed similar rates of cellproliferation relative to controls suggesting differential specificityfor different cellular events (FIG. 7C). The results demonstrate thatthymosin 815, which is upregulated in the highly motile AT3.1 and AT6.1Dunning tumor cell lines, is a positive regulator of cell motility whichis an important component of cancer metastasis.

[0100] Immunohistochemical detection of Tβ15 in prostate carcinoma

[0101] A polyclonal antibody was raised against a peptide representingthe 11 C-terminal amino acids of thymosin,β15 Synthesized peptide wascoupled with a carrier, keyhole limpet hemocyanin (KLH), and injectedinto rabbits. Antiserum was affinity-purified over the C-terminalpeptide coupled CNBr-activated sepharose 48 column. To test the 20specificity of the purified antibody, we performed Western analysis ofthe GST/thymosin v fusion proteins with the affinity-purified antiC-terminal antibody. The purified antibody strongly reacted withGST-thymosin β15 fusion protein, but did not cross react withGST-thymosin β4, and not with GST alone (FIG. 8) showing itsspecificity.

[0102] We used the affinity purified polyclonal thymosin β15 antibodyfor immunohistochemical study of human prostate carcinoma. The resultsare summarized below in Table 1. The thymosin β15 immunostaining wasobserved in the cytoplasms of epithelial cells in neoplastic prostatesbut not in normal prostates and not in the stromal cells (FIG. 10A,large arrow). Among the investigated malignant epithelia, the poorlydifferentiated prostate carcinomas displayed the most extensive andintense thymosin β15 immunoreaction (FIG. 10C), followed by moderatelydifferentiated prostate carcinomas in which not all carcinomas expressedthymosin p15 showing partial positivity (FIG. 106). In some cases,high-grade prostatic intraepithelial neoplasia (PIN) showed thymosin β15immunostaining, but to a lesser extent FIG. 10A, small arrow). In poorlydifferentiated invasive carcinoma, single cells invading stromadisplayed intense staining (FIG. 10D). The expression of thymosin β15well correlated with Gleason grade of prostate carcinoma. TABLE 1THYMOSIN β15 EXPRESSION IN HUMAN PROSTATE CARCINOMA Prostate No.Negative^(a) Partial^(b) Positive^(c) BPH 2 2 0 0 Ca Gleason 2˜5  5 3 20 Ca Gleason 6˜8  25 4 7 14 Ca Gleason 5˜10 6 0 1 5 Ca (with met) 3 0 12

[0103] Expression of thymosin β15 mRNA in mouse lung carcinoma Todetermine whether thymosin β15 may be expressed in other kind of cancercells, we tested mouse lung carcinoma cell lines by Northern analysis.The results showed the thymosin β15 expression in metastatic cell linesM27 and H59, but showed no expression in a nonmetastatic cell line LA-4(FIG. 9).

DISCUSSION

[0104] Progression to the metastatic stage is directly correlated withmortality from prostatic carcinoma. It therefore follows that the earlydiagnosis, prevention, or therapeutic treatment of metastaticprogression would lead to more effective control of this disease. TheDunning R-3327 rat prostatic adenocarcinoma model provides severalsublines with varying metastatic ability, all of which derive from anoriginal spontaneous tumor and which provide an opportunity to study thesteps leading to prostate cancer metastases (Mohler, Cancer Metast. Rev.1 2, 53-67 1993) and Pienta, et al. Cancer Surveys 1 1, 255-263 (1993)).By comparing gene expression among the Dunning cells, we cloned a novelmember of the thymosin 13 family, thymosin β15, which is expressed inhighly metastatic prostate cancer cells but not in non- or weaklymetastatic cells. The related family members thymosin β4 and β10 areexpressed equally in all of the cell lines tested such that theirexpression does not vary with increasing metastatic potential.

[0105] Thymosin β15 binds G-actin and retards actin polymerization.Because the highly motile prostate cancer cell lines showed high levelexpression of thymosin β15, we tested whether thymosin β15 transfectioninto the Dunning rat carcinoma cell lines could influence cell motility.Our results show clearly that transfection of sense or antisensethymosin β15 constructs into rat prostatic carcinoma cells cansignificantly modulate stimulated cell migration, a property notpreviously associated with β-thymosins. In cancer, the enhanced movementof malignant tumor cells through connective tissues is a majorcontributor to progression toward the metastatic stage. In order tometastasize, a tumor cell must initially dissociate from the primarytumor, migrate through connective tissue and capillary walls into thecirculatory system, and migrate again across the vascular wall into asecondary site. Therefore, increases in thymosin β15 expression inmalignant prostate carcinoma cells are believed to mediate an importantchange in tumor progression toward metastasis and that the expression ofthymosin β15 is a useful marker for diagnosis and prognosis of cancermalignancy.

[0106] Cell motility is typically associated with coordinateddisassembly and reformation of the cortical actin network (Cunningham,et al., Science 251, 1233-1236 (1991), Haugwitz, et al., Cell 79,303-314 (1994) and Stossel, Science 260, 1086-1094 (1993)) . Enhancedexpression or activation of thymosin's actin binding function maytherefore stimulate motility by enhancing the depolymerization phase ofthis process. The finding that a molecule which acts to retard actinpolymerization may stimulate cell motility is consistent with the recentfinding of Hug et al. (Hug, et al., Cell 81, 591-600 (1995) which showedthat over expression of an action capping protein in Dictyostelium cellsled to an increased rate of cell motility. The findings on therelationship between actin depolymenzation and increased motility alsosupport our hypothesis that the upregulation of thymosin 62 15 mayrepresent an important step in the progression of prostatic carcinoma tothe metastatic state. The finding that thymosin β15, which isupregulated in more highly metastatic rat prostate cancer cell lines, isalso upregulated in human prostate cancer is intriguing. At present, thebest markers for prostate cancer, such as PSA expression, are mostuseful for early detection of prostate cancer. However, they do notallow any distinction of metastatic tumor from non-metastatic tumors.

[0107] This invention has been described in detail including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of this disclosure, maymake modifications and improvements thereon without departing from thespirit and scope of the invention as set forth in the claims.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:13 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 412 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE:(ix) FEATURE: (A) NAME/KEY: Coding Sequence (B) LOCATION: 98...232 (D)OTHER INFORMATION: Exon 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:TATCAGCTAG TGGCTGCACC CGCGAACACC ACCCTGGTCC GGAGTAGCTG CGGACAGAAT 60TGCTGGCCTA GTAGAAGCTT TGGAACGAGC AGTCAAG ATG AGT GAT AAA CCA GAC 115 MetSer Asp Lys Pro Asp 1 5 TTA TCA GAA GTT GAA ACA TTT GAC AAA TCA AAG TTGAAG AAG ACT AAT 163 Leu Ser Glu Val Glu Thr Phe Asp Lys Ser Lys Leu LysLys Thr Asn 10 15 20 ACT GAA GAA AAG AAT ACT CTT CCT TCG AAG GAA ACT ATCCAG CAG GAG 211 Thr Glu Glu Lys Asn Thr Leu Pro Ser Lys Glu Thr Ile GlnGln Glu 25 30 35 AAA GAA TAT AAT CAA AGA TC ATAAAATGAG ATTCTCCTCTCAAGAGCAAC TTCAAC 267 Lys Glu Tyr Asn Gln Arg Ser 40 45 TTTGCTGGATAGTCTTGGAT TTAGACATGT TTCTGTAAAC CTATCCAATA TGTAGACATT 327 TTAGGCGGTTCCTGATAGGT TCTTAAGTAC CCTGACTGAA AGGTCAGCAT TTAACACCAA 387 TCATTAAATGTGTTTTCCAC TGCTC 412 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal(vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met SerAsp Lys Pro Asp Leu Ser Glu Val Glu Thr Phe Asp Lys Ser 1 5 10 15 LysLeu Lys Lys Thr Asn Thr Glu Glu Lys Asn Thr Leu Pro Ser Lys 20 25 30 GluThr Ile Gln Gln Glu Lys Glu Tyr Asn Gln Arg Ser 35 40 45 (2) INFORMATIONFOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 3: AGGGAACGAG 10 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENTTYPE: N-terminal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 4: Pro Lys Lys Lys Arg Lys Val 1 5 (2) INFORMATION FOR SEQ ID NO: 5:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:N-terminal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Pro Glu Lys Lys Ile Lys Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:N-terminal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Gln Pro Lys Lys Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:N-terminal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Arg Lys Lys Arg 1 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TATCAGCTAGTGGCTGCACC CGCG 24 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AAATGCTGACCTTTCAGTCA GGGT 24 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ACTCTCAATTCCACCATCTC CCAC 24 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCCTCTGAGCAGATCGTCTC TCCTTG 26 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATAATATCCCTGGGCAAACC GGTG 24 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GAGTGGAGTACCTGGAGCGC GAGC 24

What is claimed is:
 1. An isolated antibody or antibody fragment whichselectively binds human thymosin P15.
 2. The antibody fragment of claim1, wherein said fragment is a Fab, Fab′, F(ab′)2 or Fv fragment.
 3. Theantibody of claim 1, wherein said antibody is a single chain antibody.4. The antibody of claim 1, wherein said antibody is humanized.
 5. Theantibody or antibody fragment of claim 1, wherein said antibody orantibody fragment is detectably labelled.
 6. An isolated and purifiedhuman thymosin β15 having the amino acid sequence set forth in SEQ IDNO.:
 2. 7. An isolated and purified polypeptide comprising a peptideselected from the group consisting of amino acid 7 to 12 of SEQ ID NO:2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ IDNO:
 2. 8. An isolated polynucleotide encoding human thymosin β15comprising the amino acid sequence as set forth in SEQ ID NO:2.
 9. Anisolated polynucleotide encoding a polypeptide comprising a peptideselected from the group consisting of amino acid 7 to 1 2 of SEQ ID NO:2. amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 39 to 44 of SEQ IDNO:
 2. 10. The polynuc!eotide of claims 8 or 9 wherein thepolynucieotide is DNA.
 11. The polynucleotide of claims 8 or 9 whereinthe polynucleotide is cDNA.
 12. The polynucleotide of claims 8 or 9wherein the polynucleotide is RNA.
 13. An isolated polynucleotide havingthe nucleotide sequence of SEQ ID NO:1, or the complement thereto. 14.An isolated polynucleotide encoding human thymosin .β15 having thenucleotide sequence of nucleotides 98-232 of SEQ ID NO: 1, or thecomplement thereto.
 15. A recombinant vector containing the DNA of claim13 or
 14. 16. A host cell containing the vector of claim
 15. 17. Amethod of treating a neoplastic cell expressing human thymosin β15comprising, administering to the cell an effective amount of a compoundwhich suppresses the activity or production of the human thymosin β1518. The method of claim 17, wherein the compound interferes with theexpression of the human thymosin β15 gene.
 19. The method of claim 18,wherein expression of the gene is inhibited by administering antisenseoligonucleotides.
 20. The method of claim 17, wherein the compound is anantibody or fragment thereof or a single chain antibody.
 21. An isolatednucleotide segment comprising at least 10 nucleotides and hybridizesunder stringent conditions to a DNA fragment having the nucleotidesequence set forth in SEQ ID NO:1.